Purpose Recurrence in early-stage laryngeal carcinoma after radiation therapy often necessitates a total salvage laryngectomy (TSL). Understanding the genetic landscape that influences cancer progression after radiation therapy may help future treatments. We investigated early-stage laryngeal carcinoma patients treated with radiotherapy to identify mutation patterns that predispose to disease recurrence. Methods The patient cohort had 35 early-stage laryngeal head and neck squamous cell carcinoma (HNSCC) patients (T1=15, T2=20,) treated with radiotherapy (RT). We stratified patients: Responders (no recurrence, N=14), non-responders (recurrence within 12 months, N=21), and non-responders undergoing TSL (N=18). We employed whole exome sequencing to characterize gene mutations using the Genome Analysis Toolkit (GATK) for variant detection and impact on critical biological pathways and post-radiotherapy patient outcomes. Pathway analysis Ingenuity and Reactome Pathway Analysis tools were used to explore the mutated gene pathways. Results – Differential mutation analysis was performed in the respective groups to find driver genes. In the pre-treatment samples, KCNT2 and AGAP6 mutations were exclusively found in non-responders (OR=0, P=0.005 and OR=0, P=0.027, respectively), while ADAMTS7 mutations were solely present in responders (OR=inf, P=0.019). PLEC mutations were more prevalent in responders (OR=11.6, P=0.006). Pathway analysis revealed that significant genes were involved in the RND2 GTPase cycle, protein O-glycosylation-related diseases, and apoptotic pathways. Post-treatment analysis in patients undergoing TSL had enrichment of mutations in apoptosis regulation pathways. Conclusions – The study reveals that mutations in apoptosis controlling genes were predominantly represented in the larynx non-responder patients both in the pre-radiotherapy and post-TSL populations.

For patients with locally advanced, p16-negative head and neck squamous cell carcinoma (HNSCC), overall survival remains poor due to primary locoregional failure and distant metastasis following curative therapy. We aimed to understand how MAPKAPK2 (MK2) regulates HNSCC tumor cell migration and invasion, important first steps in cancer metastases. The TCGA database and HNSCC tissue microarrays were used to show that MK2 expression was associated with more advanced cancers and faster cancer recurrence rates. We observed that silencing of tumor MK2 in human cell lines (shRNA) caused a significant reduction in tumor cell migration-invasion in a complex HNSCC microphysiologic system used to recapitulate the tumor microenvironment. Murine cells (Ly2) with MK2 silenced (CRISPR-Cas9) also demonstrated reduced migration and invasion using 2D and 3D monoculture cell migration-invasions assays. Ly2 cells are orthotopic p16-negative murine metastatic cells that spontaneously metastasize, and we observed that MK2 inhibition via genetic (Cas9/CRISPR) or pharmacologic (PF-3644022) methods led to a significant reduction in the number of circulating tumor cells, fewer lymph node and lung metastases, and MK2 inhibited mice showed improved overall survival. Our findings suggest that HNSCC MK2 regulates tumor cell migration-invasion and may be a promising therapeutic target to reduce metastases.

Abstract Introduction: HPV mediated head and neck squamous cell carcinoma (HNSCC) has an immunosuppressive microenvironment which contributes to local and distant failures. We have previously shown that MAPKAPK2 (MK2) inhibition (MK2i) in HNSCC results in improved response to irradiation (RT). MK2i was synergistic with RT resulting in better control than monotherapy. To understand the mechanism behind the improvement of RT in MK2i treated mice we tested the hypothesis that MK2 ablation can enhance the radiotherapy mediated immunogenic response in a solid tumor. Methods: We used CRISPR gene editing to knockout the MK2 gene in the highly metastatic murine cell line, MLM3 (KO). We implanted tumors into c57bl6 and NSG mice and compared tumor growth compared to wild type (WT) with and without radiation. At end point, tumors were excised, digested and immunophenotyped utilizing labelled antibodies and flow cytometry. Before immunophenotyping, 8 mgs of tumor were cut off and incubated in media for 24 hours to assess cytokine release by cytokine array analysis. Results: We found that the differences in tumor growth between WT and KO cells in c57bl6 mice were significantly greater than those seen in NSG mice. Survival studies with c57bl6 mice implanted with WT or KO tumors with and without RT showed that MLM3 KO implanted mice had significantly greater survival (58.5 days) compared to WT (35.5 days). WT tumors treated with RT improved median overall survival with the WT + RT group surviving longer than WT (56 days) and the MK2 KO + RT had the longest survival overall (65 days). Immunophenotyping WT v KO tumors grown in c57bl6 mice showed an increase in CD45 cells in the KO tumors compared to WT. The cells that were increased in number in the KO tumors were labelled by Ly6G (neutrophils), F4/80 (macrophages), CD14+MD11c (dendritic cells), CD4 (Th), CD8 (Tc), CD3+NK1.1 (NKt) and NK1.1 (NK cells). Irradiating the tumors accentuated the influx of immune cells causing a greater influx of CD45 cells at 3 days post-RT which included increases in F4/80+CD80 (M1 macrophages), CD14 (monocytes), CD11c+MHCII, NK1.1, NK1.1+CD3 and CD4 cells. However, the influx of CD8 cells were reduced at 3 days. At 8 days post RT, many cell types remained elevated in the KO tumors compared to wildtype and there was a partial recovery of CD8 cell influx. However, there was also an increase in immune suppressor cells into the tumors including Ly6C+arginase (M-MDSCs), Ly6C+arginase (PMN-MDSCs) and CD4+T-bet (Th1) cells. The cytokine analysis showed that six cytokines were differentially secreted from tumors: Dkk-1, IL-2, IGFBP-5, IL-3, IL-17a and CD14. All these cytokines were suppressed in the KO with respect to the WT tumors. Conclusion: Here we show evidence that genetic ablation of MK2 in a mouse model of HPV mediated HNSCC results in an increase in immune cells infiltrate into tumors. Irradiation enhances this difference by stimulating a greater influx of immune cells. These data suggest that tumoral MK2 may aid in the immunosuppression of tumors in HNSCC. Citation Format: Deri Morgan, Colby Spiess, Alyssa Schmidt, Dakota D.D. Okuwone, Harmony Saunders, Chris E. Lominska, Mary A. Markiewicz, Yelder Tonia, Devin Shrock, Yuting Lin, Hao Gao, Gregory N Gan. MK2 knockout and radiation enhances immune cell influx in a mouse model of HPV+ head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C009.

Abstract Background: Head and neck squamous carcinoma (HNSCC) results in poor patient outcomes driven primarily by locoregional and distant metastatic tumor spread. We have demonstrated that highly phosphorylated/active MAPKAPK-2 (MK2) is a poor prognostic factor in HNSCC patients. MK2 is associated with the expression of pro-inflammatory cytokines that are linked with creating a pro-tumorigenic microenvironment. Nevertheless, how MK2 contributes to HNSCC development has not been established. In this study, we aimed to identify the effect of MK2 on cancer progression, immune infiltration, and cytokine secretion. Method: To assess the effect of tumor MK2 in vivo, we used CRISPR-Cas9 to knock out (KO) MK2 in a metastatic murine HNSCC cell line, Ly2, and orthotopically implanted wildtype (WT) or MK2 KO cells into the floor-of-mouth of Balb/c mice. At the endpoint of the experiment, the tumors were excised, and myeloid and lymphocyte immune profiling was done via flow cytometry. Mouse lungs and cervical lymph nodes were dissected, paraffin-fixed, sectioned, and H&E stained, and the presence/quantity of metastatic foci was evaluated by a blinded board-certified pathologist. We broadly evaluated the tumor secretome via a 105-cytokine dot blot array using 8 mg of the same resected tumor sample and performed densitometric analysis comparing WT and KO tumors. Results: Loss of MK2 significantly reduced primary tumor growth, as well as lymph node and lung metastasis, compared to WT tumors. Tumor immunophenotyping by flow cytometry revealed that MK2 KO tumors had increased overall immune cell (CD45+) infiltration, but decreased quantities of immunosuppressive neutrophils (CD11b+Ly6G+Arg1+Ly6C-) and a shift in macrophage polarity favoring M1 (anti-tumor) macrophages (CD11b+Ly6G-F4-80+CD80+). In addition, there were increased quantities of anti-tumor immune cells including natural killer (NK)-T, CD4+, and CD8+ T cells in the MK2-KO tumors, while the proportion of CD4+CD25+ Tregs and exhausted CD8 T cells (CD8+PD1+) was decreased compared to WT tumors. Furthermore, cytokine array analysis revealed that secreted levels of several cytokines, including CXCL1, CXCL2, GM-CSF, G-CSF, IL-1alpha, and IL-6, were significantly lower in MK2 KO tumors compared to WT tumors. Conclusion: These results show that tumoral MK2 signaling promotes tumor growth, metastasis, immunosuppressive inflammation, and pro-tumorigenic cytokine release which substantiates future therapeutic targeting of this pathway. Citation Format: Dakota D.D. Okwuone, Deri Morgan, Alyssa Schmidt, Devin Shrock, Yuting Lin, Hao Gao, Sufi M. Thomas, Gregory N. Gan. Tumor MK2 drives tumor progression and an immunosuppressive microenvironment in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C010.

Head and neck cancer (HNC) is a challenging disease that lacks effective treatment, particularly in the cases that spread locoregionally and metastasize distantly, dramatically reducing patient survival rates. Expanding the understanding of the mechanisms of the metastatic cascade is critical for creating more effective therapeutics that improve outcomes for HNC patients. A true grasp of cancer metastasis requires the consideration of all cell types that contribute to the inflammatory HNC microenvironment as drivers of this process. More emphasis now is being placed on exploring the roles of the different immune cells in cancer control, tumorigenesis and metastasis. Myeloid cells are the most numerous immune cell types in the body, and they are actively recruited and reprogrammed by tumor cells to behave in a variety of ways. These cells are remarkably diverse in phenotype and function, and the part they play in tumor spread greatly differs based on the cell type. This review will focus on summarizing the roles of macrophages, neutrophils, myeloid derived suppressor cells (MDSCs), and dendritic cells (DCs) in driving HNC metastasis by examining the current knowledge base and offering potential new routes through which to target and treat this deadly process.

Background: Bladder cancer patients unable to receive cystectomy or who choose to pursue organ-sparing approach are managed with definitive (chemo)radiotherapy. However, this standard of care has not evolved in decades and disease recurrence and survival outcomes remain poor. Identifying novel therapies to combine with radiotherapy (RT) is therefore paramount to improve overall patient outcomes and survival. One approach is to find cellular mechanisms that can be targeted to increase the radiosensitivity of bladder cancer. The stress- activated kinase directly downstream from p38 mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase activated protein kinase 2 (MAPKAPK2 or MK2), has been shown to enhance cancer-mediated inflammation, mesenchymal gene expression, and in vivo tumor growth. Here we examined the impact that MK2 knockdown (KD) has on bladder cancer cell radiosensitivity. Methods: We utilized short hairpin RNA (shRNA) KD of MK2 using lentiviral transfection in the bladder cancer cell lines, T24 and HTB9. We compared the growth of KD cells to wild type using colony formation assays, proliferation assays and cell counts to determine differences in cell growth. Apoptosis was examined by annexin-based flow cytometry and western blots. Flow cytometry was also used for cell cycle analysis. Results: KD clones showed a greater than 90% inhibition of MK2 expression as determined by western blot. Clonogenic assays exhibited an increase in radiosensitivity among the MK2 KD bladder cancer cells. These data were supported with proliferation assays that displayed a greater reduction in cell number following RT in MK2 KD bladder cancer cells. Annexin V binding in bladder cancer cells suggested increased apoptosis in MK2 KD cells. This was confirmed by comparing the amount of cleaved caspase products for the caspases and 8 to scrambled control (SCR), and the release of cytochrome C into the cytosol. Both cell types showed disruptions in the cell cycle but at different points in the cycle. Conclusion: These results show that MK2 controls irradiation-induced apoptosis in bladder cancer cells.

Abstract Our previous work has shown that the phosphorylation of MK2, a downstream kinase of the p38 pathway, is a prognostic feature of head and neck cancer (HNC) where patients with higher MK2 phosphorylation had lower overall survival. Studies with a MK2 inhibitor demonstrate control of HNC growth in a mouse model that was synergistic with radiotherapy (RT) in inhibiting tumor growth. The mechanism of action that MK2 inhibition combined with RT has on modulating HNC growth is unknown. A better understanding of this mechanism might reveal how to effectively deploy MK2 inhibitors in HNC treatment. Here we describe our studies in which we suppressed MK2 expression in HNC cell lines and found increased genomic instability in response to radiotherapy. Tu167 and Cal27 HNC cells were transfected with a stable shRNA construct that suppressed ~90% of the MK2 expression compared to a control shRNA construct (SCR) as determined by immunoblot. Loss of MK2 did not significantly affect cell growth nor did it affect cells from forming colonies. However, exposing the cells to 10 Gy radiation caused the formation of micronuclei that was increased in the shRNA cells compared to SCR at 48 hours (Cal 27 SCR + 10 Gy = 0.5 ± 0.3 Micronuclei/cell v Cal27 shRNA + 10 Gy = 1.6 ± 0.1. Micronuclei/cell P<0.01; Tu167 SCR + 10 Gy = 1.1 ± 0.2 Micronuclei/cell; Tu167 shRNA + 10 Gy = 2.3 ± .3; Micronuclei/cell p<0.001). Immunocytochemical techniques were used to image cells at 60x magnification. In conjunction with increased micronuclei formation, we saw an increase expression in both the DNA sensor cGAS, and phosphorylation of STING in the shRNA cells compared to SCR. The increase in micronuclei caused by radiation was largely inhibited by cytochalasin B which also inhibited the increase in STING phosphorylation. There was no difference in the staining of γH2AX after 2 hours following RT but after 48 hours the γH2AX staining was concentrated in the micronuclei which were more numerous in the shRNA cells. These data provide evidence that MK2 plays a role in controlling the DNA damage response, and we show for the first time that loss of MK2 increases the number of micronuclei formed causing an enhanced activation of the cGAS-STING pathway, an innate immune DNA sensing pathway. This suggests that inhibition of MK2 may enhance RT effectiveness by activating STING and facilitating an anti-tumor immune response. Citation Format: Colby Spiess, Grace Millington, Hannah Smith, Dakota D. Okwuone, Kiersten Berggren, Deri Morgan, Chris Lominska, Sufi M. Thomas, Jingxin Wang, Mary Markiewicz, Gregory N. Gan. Loss of head and neck squamous cell carcinoma MK2 increases STING activation through increased micronuclei formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 302.

Poor 5-year overall survival in head and neck cancer (HNC) can be attributed to high rates of locoregional and distant metastasis. We have previously demonstrated that high phosphorylation/activation of MAPK-activated protein kinase 2 (MK2), a stress-activated kinase directly downstream of p38 MAPK, in head and neck squamous cell carcinoma (HNSCC) is associated with worse overall survival and inhibition of the MK2 pathway enhances in vivo HNSCC radiosensitivity. Our prior work also demonstrates that radiotherapy-induced epithelial-to-mesenchymal transition (EMT) gene expression can be suppressed in cells when MK2 is inhibited. Several studies have shown that EMT plays a prominent role in HNSCC tumor invasion, treatment resistance, and locoregional metastases to lymph nodes. In this study, we investigate the impact of MK2 activity on HNSCC tumor progression by examining its contribution to cancer cell morphology. We used lentiviral-shRNA and the CRISPR-Cas9 system to knock down/out the MK2 gene in multiple human and murine HNSCC cell lines. We measured tumor cell migration and invasion in both classic 2D in vitro culture as well as with 3D spheroids. We performed unbiased analysis via RNAseq in human HNSCC cell lines comparing MK2 shRNA vs scramble cell lines. Substantial gene and pathway alterations were examined using a combination of immunoblot and RT-qPCR. Our data shows knocking down/out MK2 in HNSCC cells significantly diminishes cell migration and invasion in 2D and 3D in vitro culture assays. RNAseq analysis demonstrated loss of MK2 leads to a significant increase in many cell adhesion molecules, including E-cadherin, and a decrease in N-cadherin, suggesting a shift from mesenchymal to more epithelial phenotype. Furthermore, we note expression of multiple matrix metalloproteinases and EMT markers (i.e., Snail) are substantially reduced in MK2 shRNA and KO cell lines. These results indicate that the MK2 pathway is involved in HNSCC motility and invasion. We also observe the loss of MK2 contributes to a shift in the epithelial:mesenchymal ratio potentially by suppressing cancer EMT plasticity. All in all, this study provides evidence suggesting tumor MK2 activation mediates HNC progression. Citation Format: Dakota D. Okwuone, Deri Morgan, Hannah M. Smith, Grace Millington, Kiersten L. Berggren, Christopher E. Lominska, Sufi M. Thomas, Gregory N. Gan. Tumor MK2 signaling regulates cell migration and invasion in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3601.

HNSCC tumor MK2 inhibition enhances RT-mediated micronuclei formation and subsequent cGAS-STING-IFNβ1 levels. Loss of HNSCC MK2 leads to increased CD4-CD8 T-cell infiltration into the tumor and this effect is enhanced following RT. Targeting tumor MK2 may facilitate improved tumor control.

Voltage‐gated ion channels, whose first identified function was to generate action potentials, are divided into subfamilies with numerous members. The family of voltage‐gated proton channels (H V ) is tiny. To date, all species found to express H V have exclusively one gene that codes for this unique ion channel. Here we report the discovery and characterization of three proton channel genes in the classical model system of neural plasticity, Aplysia californica . The three channels (AcH V 1, AcH V 2, and AcH V 3) are distributed throughout the whole animal. Patch‐clamp analysis confirmed proton selectivity of these channels but they all differed markedly in gating. AcH V 1 gating resembled H V in mammalian cells where it is responsible for proton extrusion and charge compensation. AcH V 2 activates more negatively and conducts extensive inward proton current, properties likely to acidify the cytosol. AcH V 3, which differs from AcH V 1 and AcH V 2 in lacking the first arginine in the S4 helix, exhibits proton selective leak currents and weak voltage dependence. We report the expansion of the proton channel family, demonstrating for the first time the expression of three functionally distinct proton channels in a single species.

Cancer and the immune system share an intimate relationship. Chronic inflammation increases the risk of cancer occurrence and can also drive inflammatory mediators into the tumor microenvironment enhancing tumor growth and survival. The p38 MAPK pathway is activated both acutely and chronically by stress, inflammatory chemokines, chronic inflammatory conditions, and cancer. These properties have led to extensive efforts to find effective drugs targeting p38, which have been unsuccessful. The immediate downstream serine/threonine kinase and substrate of p38 MAPK, mitogen-activated-protein-kinase-activated-protein-kinase-2 (MK2) protects cells against stressors by regulating the DNA damage response, transcription, protein and messenger RNA stability, and motility. The phosphorylation of downstream substrates by MK2 increases inflammatory cytokine production, drives an immune response, and contributes to wound healing. By binding directly to p38 MAPK, MK2 is responsible for the export of p38 MAPK from the nucleus which gives MK2 properties that make it unique among the large number of p38 MAPK substrates. Many of the substrates of both p38 MAPK and MK2 are separated between the cytosol and nucleus and interfering with MK2 and altering this intracellular translocation has implications for the actions of both p38 MAPK and MK2. The inhibition of MK2 has shown promise in combination with both chemotherapy and radiotherapy as a method for controlling cancer growth and metastasis in a variety of cancers. Whereas the current data are encouraging the field requires the development of selective and well tolerated drugs to target MK2 and a better understanding of its effects for effective clinical use.

We have previously identified the p38 substrate MAPK-activated protein kinase-2 (MK2) as a radiation response stress pathway regulating inflammatory cytokine production that facilitates EMT pathway activation and tumor growth in head and neck cancer. Here we examine whether MK2 knockdown in bladder cancer cells alters their ability to produce immune mediators and to resist radiation.In order to study the impact of MK2 in bladder cancer (i.e., T24, HTB9), we generated both scrambled control (SCR) and MK2 shRNA using a lentiviral transduction system and selected for clones via puromycin selection. Generally, all MK2 shRNA clones demonstrated >88% reduction in MK2 protein expression by immunoblot. Increased phosphorylation of MK2 (p272) compared to control was detected 48 hours after the exposure of cells to 10 Gy radiation which was not seen in shRNA cells. Gene expression analysis of the bladder cells at 48 or 72 hours after 10 Gy radiation showed an increase in the expression of the inflammatory cytokines IL-6 and TNFα and genes that regulate epithelial to mesenchymal transition; SNAI1 and vimentin. A reduced expression of these genes was seen in the shRNA cells following radiation demonstrating MK2 as an important signaling molecule in this response. We then examined whether MK2 also impacted tumor proliferation, radiosensitivity and the cell cycle. The proliferation rates of shRNA cells compared to SCR cells showed no difference under control conditions in both cell lines. However, in response to radiation both T24 and HTB9 shRNA cells showed a greater loss of cells compared to SCR as determined by CyQUANT DNA fluorescence at 24, 48, 72 and 144 hours. Furthermore, shRNA cells showed reduced clonogenic activity after radiation compared to SCR as determined by colony-forming assays using 0-10 Gy doses in 2 Gy increments. Analysis of cell cycle changes induced by radiation at 28 hours showed a pronounced shift of HTB9 cells from 57.8 (% G1 Phase), 19.1 (% S phase) and 22.1 (% G2/M) phase to 7.6, 3.4, 86.7. This pattern was significantly different in shRNA cells that showed proportions of 46.2, 17.8, 32.0 without radiation which shifted to 20.6, 5.9 and 67.3 28 hours after irradiation. The percentage of cells in G1, and G2/M phase 28 hours after irradiation was significantly different between SCR and shRNA (p<0.001) suggesting that MK2 knockdown altered radiosensitivity by impacting cell cycle kinetics.In summary, we measured a significant increase in the sensitivity of bladder cancer cells to radiation following knockdown of MK2. The data suggests a reduction in survival of tumors cells exposed to radiation with MK2 knockdown through cell cycle changes and reduced production of survival and growth signals. Citation Format: Deri Morgan, Grace Millington, Hanna Smith, Colby Spiess, Kiersten L. Berggren, Xinglei Shen, Gregory N. Gan. Knockdown of MK2 in bladder cancer cells increases their radiosensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 792.

Approximately 20% of breast cancers are HER2-positive. Trastuzumab has improved patient outcomes significantly for these cancers. However, acquired resistance remains a major hurdle in the clinical management of these patients. Therefore, identifying molecular changes that cause trastuzumab resistance is worthwhile. STAT6 is a transcription factor that regulates a variety of genes involved in cell cycle regulation, growth inhibition, and apoptosis. STAT6 expression is lost in approximately 3% of breast cancers, but little work has been done in the context of trastuzumab resistance in breast cancer. In isogenic cell line pairs, we observed that trastuzumab-resistant cells expressed significantly lower levels of STAT6 compared to trastuzumab-sensitive cells. Therefore, in order to study the consequences of STAT6 loss in HER2+ breast cancer, we knocked out both alleles of the STAT6 gene using somatic cell gene targeting. Interestingly, loss of STAT6 resulted in anchorage-independent growth and changes in several genes involved in epithelial to mesenchymal transition. This study suggests that STAT6 may play a role in the pathophysiology of HER2+ human breast cancer.

Expression of the voltage gated proton channel (H(v)1) as identified by immunocytochemistry has been reported previously in breast cancer tissue. Increased expression of H(V)1 was correlated with poor prognosis and decreased overall and disease-free survival but the mechanism of its involvement in the disease is unknown. Here we present electrophysiological recordings of H(V)1 channel activity, confirming its presence and function in the plasma membrane of a breast cancer cell line, MDA-MB-231. With western blotting we identify significant levels of H(V)1 expression in 3 out of 8 "triple negative" breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of H(V)1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of H(V)1 using shRNA (knock-down; KD) and by eliminating H(V)1 using CRISPR/Cas9 gene editing (knock-out; KO). Surprisingly, these two approaches produced incongruous effects. Knock-down of H(V)1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H2O2 release. In contrast, H(V)1 Knock-out cells did not show reduced migration or H2O2 release. H(V)1 KO but not KD cells showed an increased glycolytic rate accompanied by an increase in p-AKT (phospho-AKT, Ser473) activity. The expression of CD171/LCAM-1, an adhesion molecule and prognostic indicator for breast cancer, was reduced in H(V)1 KO cells. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from H(V)1 KO cells grew less than WT in mass, with lower staining for the Ki-67 marker for cell proliferation rate. Therefore, deletion of H(V)1 expression in MDA-MB-231 cells limits tumor growth rate. The limited growth thus appears to be independent of oxidant production by NADPH oxidase molecules and to be mediated by cell adhesion molecules. Although H(V)1 KO and KD affect certain cellular mechanisms differently, both implicate H(V)1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.

The voltage-gated proton channel (H(v)1) is a voltage sensor that also conducts protons. The singular ability of protons to penetrate proteins complicates distinguishing closed and open channels. When we replaced valine with histidine at position 116 in the external vestibule of hH(v)1, current was potently inhibited by externally applied Zn2+ in a construct lacking the two His that bind Zn2+. in WT channels. High-affinity binding with profound effects at 10 nM Zn2+ at pH(o) 7 suggests additional groups contribute. We hypothesized that Asp(185), which faces position 116 in our closed-state model, contributes to Zn2+. chelation. Confirming this prediction, V116H/D185N abolished Zn2+. binding. Studied in a C-terminal truncated monomeric construct, V116H channels activated rapidly. Anomalously, Zn2+ slowed activation, producing a time constant independent of both voltage and Zn2+. concentration. We hypothesized that slow turn-on of H+. current in the presence of Zn2+ reflects the rate of Zn2+ unbinding from the channel, analogous to drug-receptor dissociation reactions. This behavior in turn suggests that the affinity for Zn2+ is greater in the closed state of hH(v)1. Supporting this hypothesis, pulse pairs revealed a rapid component of activation whose amplitude decreased after longer intervals at negative voltages as closed channels bound Zn2+. The lower affinity of Zn2+ in open channels is consistent with the idea that structural rearrangements within the transmembrane region bring Arg(205) near position 116, electrostatically expelling Zn2+. This phenomenon provides direct evidence that Asp(185) opposes position 116 in closed channels and that Arg(205) moves between them when the channel opens.

The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this "gating pore" when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hHV1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open-closed gating, but strikingly, at negative voltages where "normal" gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 μM Zn2+ Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.

A large family of membrane proteins, the voltage gated ion channels, regulate a vast array of physiological functions in essentially all life forms. The mechanism by which these molecules sense membrane potential and respond by creating ionic conduction is incompletely understood. These channels contain a ring of hydrophobic amino acids near the center of the voltage sensing domain in the membrane, the “hydrophobic gasket,” HG, which is anchored by a highly conserved Phe (part of the Charge Transfer Center) in nearly all voltage-gated ion channels and voltage-sensing phosphatases. Various functions of this structure have been suggested in addition to its evident role of separating internal and external aqueous solutions. During gating, the periodic cationic residues in the S4 helix are thought to ratchet past the HG. Here we identify the HG in voltage-gated proton channels and test the hypothesis that it functions as a secondary selectivity filter. Selectivity is ensured primarily by the interaction of Asp112 and one or more Arg in S4. However, molecular dynamics simulations indicate that cation exclusion may not be complete at the Asp-Arg selectivity filter. Potential of mean-force calculations show that the top of the free energy barrier opposing Na+ permeation coincides with the most hydrophobic region of the pore, corresponding with the HG region in other voltage-sensing domains. We replaced amino acids of the HG with less hydrophobic or hydrophilic ones, both individually and in groups, and measured reversal potentials at various pH and in the presence of Na+. All mutants tested were selective for H+ and did not conduct Na+, with PH/PNa > 107 as a lower limit. We conclude that proton selectivity is accomplished by the primary selectivity filter, comprising the interaction of Asp and Arg.

Voltage-gated proton channels, HV1, were first reported in Helix aspersa snail neurons. These H+ channels open very rapidly, two to three orders of magnitude faster than mammalian HV1. Here we identify an HV1 gene in the snail Helisoma trivolvis and verify protein level expression by Western blotting of H. trivolvis brain lysate. Expressed in mammalian cells, HtHV1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hHV1 (human) at pHo 7, between 50 and 90 mV. In contrast to most HV1, activation of HtHV1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5 e0 suggests that HtHV1 functions as a dimer, evidently with highly cooperative gating. HtHV1 opening is exquisitely sensitive to pHo, whereas closing is nearly independent of pHo. Zn2+ and Cd2+ inhibit HtHV1 currents in the micromolar range, slowing activation, shifting the proton conductance–voltage (gH-V) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtHV1 possessing three of the four amino acids that coordinate Zn2+ in mammalian HV1. All known HV1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the gH-V relationship for a unit change in either pHo or pHi. This property is crucial for all the functions of HV1 in many species and numerous human cells. The HtHV1 channel exhibits normal or supernormal pHo dependence, but weak pHi dependence. Under favorable conditions, this might result in the HtHV1 channel conducting inward currents and perhaps mediating a proton action potential. The anomalous ΔpH-dependent gating of HtHV1 channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201711968).